Ozone (O3) exposure associates with exacerbation of asthma and altered lung function in adults and children, and mortality. Over 50% of the U.S. population live in regions where O3 concentrations approach or exceed the National Ambient Air Quality Standard of 0.12parts per million (ppm). We previously identified the innate immunity gene toll-like receptor 4 (Tlr4) as a candidate susceptibility gene for 03-induced lung hyperpermeability and inflammation. C3H/HeOuJ mice (Tlr4 sufficient) are significantly more susceptible to OS-induced hyperpermeability and inflammation compared to coisogenic C3H/HeJ mice (Tlr4 dominant negative mutation). However, the downstream effector mechanisms for TLR4-mediated responses are still unclear. The overall objective of this proposal is to determine the mechanisms by which TLR4 mediates OS- induced lung inflammation and hyperpermeability. The proposed studies will use a multidisciplinary approach using molecular biology, genetics, and pharmacological techniques in an in vivo animal model to address the following specific aims: 1) examine the influence of strain background on the role of TLR4 in OS-induced lung hyperpermeability and inflammation using mice that are TLR4 dominant negative and TLR4 transgenic mice; 2) determine if immediate signaling events downstream of TLR4 are altered in response to O3 exposure in Tlr4 deficient or Tlr4 over-expressed mice and to evaluate the downstream cytokine profiles for these strains to determine if strain background can modulate the types of cytokines produced; 3) investigate the downstream mechanisms by which TLR4-dependent pathways regulate OS- induced responses by verifying the importance of candidate genes identified from Affymetrix global gene arrays in vivo using molecular approaches and utilizing genetic (knockout mice) and pharmacologic methods to investigate specific candidate genes previously identified by the microarray approach. This proposal will enhance our understanding of the mechanisms of OS-induced lung inflammation and injury by the identification of novel pathways regulating OSsusceptibility. These novel pathways may provide preventive strategies for those individuals susceptible to O3.